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Page Title: Appendix I. LABORATORY PROTOCOLS FOR HepG2-EcR SCREENING ASSAY
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ERDC TN-DOER-C30
February 2003
Appendix I
LABORATORY PROTOCOLS
FOR HepG2-EcR SCREENING ASSAY
Protocol I. Cell Culture and Reagent Preparation
I.
MATERIALS AND EQUIPMENT
Vertical laminar flow hood
37 C CO2 incubator
Countertop Centrifuge
Autoclave
Ultra-pure water
Sterile filters (0.1 m), VacuCap 90 (Gelman Sciences #09730265B)
Sterile, disposable 5, 10 mL, and pasteur pipets (9", autoclaved)
Sterile T-75 (75 cm2 growth surface area) culture flasks
Sterile syringes and 0.22-m syringe filters
Squirt bottle of 70 percent reagent alcohol
Parafilm
Water bath @ 37 C
Alcohol bath
Aspirator
Rechargeable portable pipettors and tips
Inverted microscope
Isopropanol freezing chamber
Liquid nitrogen Dewar @ -195 C
HepG2-EcR (recombinant human hepatoma) cells
II.
REAGENTS
A. PRODUCT INFORMATION
Minimum Essential Medium (MEM), GIBCO BRL #51200-038
Fetal Bovine Serum (FBS), GIBCO BRL #16000-044
L-Glutamine (200 mM, 29.2 mg/ml), GIBCO BRL #25030-081
Hygromycin B (100 mg/ml), InVitrogen #55-0280
Zeocin (100 mg/ml), InVitrogen #R250-01
Phosphate Buffered Saline (PBS), GIBCO BRL #14190-144
Trypsin-EDTA (0.5-percent Trypsin, 0.53-mM EDTA), 10x, GIBCO BRL #15400-054
Trypan Blue (0.4 percent), GIBCO BRL# 15250-061
Dimethyl Sulfoxide (DMSO), SIGMA #D-2650
Cell culture handling and reagent preparation throughout the assay should be carried out only under
a sterilized laminar hood using aseptic techniques. Only the last part of the HepG2-EcR assay
("Day 4 (Assay Takedown)" in the "Performance of the Assay" section) need not be sterile.
9

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